Redox Signaling in Widespread Health Benefits of Exercise: Forum Review Article

Antioxidants and Redox Signaling
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© Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2019.7949
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Forum Review Article
REDOX SIGNALING IN WIDESPREAD HEALTH BENEFITS OF

EXERCISE
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Ruy A. LOUZADA1,2*, Jessica BOUVIERE1, Leonardo P. MATTA1, Joao Pedro S. WERNECK-DE-

CASTRO3, Corinne DUPUY2, Denise P. CARVALHO1, Rodrigo S. FORTUNATO1
1
Institut of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, 21941-
590, Brazil.
Downloaded by National University of Singapore NUS SWETS/24793917 from www.liebertpub.com at 03/16/20. For personal use only.
2
Université Paris-Sud, Orsay, UMR 8200 CNRS and Institut Gustave Roussy, 94800 Villejuif,
REDOX SIGNALING IN WIDESPREAD HEALTH BENEFITS OF EXERCISE (DOI: 10.1089/ars.2019.7949)
France
3
Division of Endocrinology, Diabetes and Metabolism, University of Miami, Miller School
of Medicine, FL33136 Miami, Florida, USA
Running title: Redox signaling in widespread effects of exercise.
Keywords: ROS; Exercise, Myokines; redox signaling;
*
Corresponding author:
Ruy A. Louzada
Laboratório de Fisiologia Endócrina Doris Rosenthal
Institut of Biophysics Carlos Chagas Filho,
Federal University of Rio de Janeiro
Brazil, 21941-590
andraderuy@hotmail.com
Word count: 5728
Reference numbers: 172
Number of color illustrations: 5 (Only online, no hardcopy)
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Abstract
Significance: Exercise-induced ROS production activates multiple intracellular signaling
pathways through genomic and non-genomic mechanisms that are responsible for the
beneficial effects of exercise in muscle. Beyond the positive effect of exercise on skeletal
muscle cells, other tissues such as white and brown adipose, liver, central nervous system,
endothelial, heart and endocrine organ tissues are also responsive to exercise.
Recent Advances: Crosstalk between different cells is essential to achieve homeostasis and
to promote the benefits of exercise through paracrine or endocrine signaling. This

crosstalk can be mediated by different effectors that include the secretion of metabolites
of muscle contraction, myokines and exosomes. During the past 20 years, it has been
demonstrated that contracting muscle cells produce and secrete different classes of
myokines, which functionally link muscle with nearly all other cell types.
Critical Issues: The redox signaling behind this exercise-induced crosstalk is now being
decoded. Many of these widespread beneficial effects of exercise require not only a
complex ROS-dependent intramuscular signaling cascade, but simultaneously, an
integrated network with many remote tissues.
Future Directions: Strong evidence suggests that the powerful beneficial effect of regular
physical activity for preventing (or treating) a large range of disorders might also rely on
ROS-mediated signaling. Within a contracting muscle, ROS signaling may control exosomes
and myokines secretion. In remote tissues, exercise generates regular and synchronized
ROS waves, creating a transient pro-oxidative environment in many cells. These new
concepts integrate exercise, ROS-mediated signaling and the widespread health benefits of
exercise.
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1. Introduction
Reactive oxygen species (ROS), such as superoxide (O2•-), hydroxyl (OH•) and the
non-free radical species, H2O2, are small radical or non-radical molecules derived from
molecular oxygen (O2) (142). Superoxide and nitric oxide seem to be the primary free
radicals generated by different sources in contracting skeletal muscle during and
immediately after exercise (84, 129, 130). ROS have a critical role as the signaling
molecules that are necessary for skeletal muscle force development and to achieve the
adaptive responses to physical exercise (64, 133). A more detailed description of how
physical exercise increases skeletal muscle ROS generation and the role of ROS-mediated
signaling for muscular exercise adaptations is provided by Bouviere et al. in this current
special forum issue, and has also been extensively reviewed by others (63, 84, 129, 130).
In 1961, the term “exercise factors” was used to define a group of factors
delivered by contracting muscles that were able to lower glycemia (62). Nowadays,
metabolites, lipid peroxidation products, extracellular ROS, myokines and exosomes are
the main suggested mechanisms through which muscle cells communicate with remote
cells. These factors that are modulated by exercise, in a chronic perspective, are directly
related to the benefits of physical exercise under different physiological and
pathophysiological conditions. In this review, we will focus on the effects of physical
exercise beyond skeletal muscle, providing an overview about the communication
between contracting muscles and remote tissues through ROS-mediated mechanisms.
2. Hypothetical model of ROS Waves
The ROS waves hypothesis is based on the ability of physical exercise to increase
ROS production in contracting muscles and its later spread to remote tissues. A
hypothetical model is proposed in figure 1 to summarize the probable mechanisms
through which contracting skeletal muscle communicates with remote tissues, leading to
changes in redox-mediated signaling pathways that operate the widespread health
beneficial effects of exercise. A first phase of ROS waves following exercise occurs in
contracting muscle and promote multiple post-translational modifications of aminoacids in
many kinases and phosphatases (e.g. AMPK, CaMK, PTEN, PPIA, etc.) that are crucial for
the local stimulus of muscular glucose uptake, mitochondrial biogenesis and antioxidant
capacity. Furthermore, skeletal muscles also produce ROS in the extracellular milieu that
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leads to macromolecules oxidation, such as lipid peroxidation. Interestingly, lipid
peroxidation products can act as signaling molecules. Myokines and exosomes might be
released from skeletal muscle in response to intracellular ROS stimulated by physical

exercise. Once in blood circulation, these mediators change the function of remote tissues
during and after exercise periods. Furthermore, the second phase of ROS waves might
stimulate many similar redox-sensible signaling pathways in non-contracting tissues that
promote widespread responses to physical exercise (Figure 1B).
In 1978, Dillard et al. found that expired pentane, an index of lipid peroxidation,
increased during aerobic physical exercise (42). One year later, Brady et al. demonstrated
that lipid peroxidation levels were higher in the skeletal muscle and liver of rats
immediately after swimming (22). Probably, this was the first indirect demonstration of
the ability of physical exercise to increase ROS generation, but the tissue involved in their
production was only revealed three years later. Davies et al. showed using electron
paramagnetic resonance that skeletal muscle and liver ROS production are increased
immediately after exhaustive exercise (36). Nowadays, it is well known that skeletal
muscle contractile activity leads to ROS generation, which is related to biomolecule
oxidation, not only in skeletal muscle, but also in other tissues (114, 130). Electrical
stimulation of the hind limb muscle in adult mice was found to increase malonaldehyde
levels in serum and liver and decreased liver glutathione and protein thiol content (34),
suggesting that the ROS generated by skeletal muscle could oxidize extracellular molecules
also in non-contracting tissues, such as the liver. In humans, the venous concentration of
oxidized glutathione was found to increase after physical exercise in an intensity-
dependent manner (35, 114).
The redox communication between contracting muscle and remote tissues can be
mediated by several mechanisms: 1) lactate conversion to pyruvate in almost every cell,
which increases NADH levels, 2) purines released by contracting muscle that serve as
substrates for xanthine oxidase (XO), 3) myokines, 4) ROS gradient reaching adjacent cells,
5) lipid peroxidation products and 6) exosomes secretion. The oxidative environment
induced in remote tissues by physical exercise is probably related to protein modification
in their cysteine residues, which cause structural and functional modifications (Figure 1)
and increased oxidation markers that are detected following exercise in non-contracting
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tissues. Moreover, several myokines released during physical exercise control the
expression of genes in a ROS-dependent manner, as well as the activity of transcription
factors, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha

(PGC-1, nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B
(NF-B) (33, 90, 123, 142).
3. Adaptations and benefits of redox signaling due to exercise
The expression of PGC-1 during physical exercise is dependent on a tightly, fine-
tuned redox-mediated signal (87), and boosting PGC-1has been related to protection
against several pathological conditions, such as sarcopenia, insulin resistance and cancer-
induced cachexia (68). PGC-1is responsible for many of the beneficial effects of exercise
in muscle due to its ability to orchestrate the transcription of both nuclear and
mitochondrial DNA, promoting a balanced expression of mitochondrial proteins and
antioxidant defences against exacerbated ROS production. Importantly, an accumulation
of PGC-1 after each bout of exercise is indispensable to achieve these beneficial effects
(47, 53, 70).
Many of the intracellular actors involved in obtaining the beneficial effects of
exercise contain redox sensible regions that are directly affected by ROS (discussed by
Bouviere et al. in this current special forum issue). The crucial requirement of optimal ROS
signaling is clearly observed by the effect of antioxidants in hampering many important
adaptations that are related to endurance training, such as an increase in exercise capacity
(21, 64), mitochondrial biogenesis (153), muscular glucose uptake, improvement in insulin
sensitivity (133) and the increase of lean mass after resistance training (17).
Several chronic exercise adaptations are dependent on ROS produced by skeletal
muscle in response to an acute exercise session. Transient increases in ROS induced
through bouts of acute exercise stimulate Nuclear factor erythroid 2 – related factor 2
(Nrf2) activation. Nrf2 is the master regulator of antioxidant defences, a transcription
factor that regulates the expression of more than 200 cytoprotective genes (44). Regular
aerobic exercise in rodent models has consistently been shown to activate Nrf2 signaling in
multiple tissues, including skeletal muscle (111), kidney (122), brain (162), liver (135) and
myocardium (145), which leads to the upregulation of endogenous antioxidant defences
and an overall greater ability to counteract the damaging effects of oxidative stress.
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Regarding protein quality control, exercise might be important to the correct
folding of proteins, due to the creation of an oxidative redox potential needed to oxidize
the free sulfhydryl groups of cysteine leading to disulphide bonds formation that stabilise
the protein conformation (166) (Figure 1). Antioxidant administration before exercise (64,
133) prevents some of the most important exercise responses, probably by maintaining
the intracellular environment in an inappropriate reduced state. This reduced-state may
impair the correct folding of born proteins (166). Loss of protein folding regulation leads
to an accumulation of unfolded or misfolded proteins inside the endoplasmic reticulum

(ER) lumen and drives ER stress (ERS), which accentuates the progression of many
diseases, including Alzheimer’s. Unlike almost all other cellular locations that have
reducing redox potentials, the typical endoplasmic reticulum has the oxidative redox
potential necessary to form disulphide bonds. It has been reported that the endoplasmic
reticulum of insulin-resistant rodents contains much higher proportions of unfolded
polypeptides and fewer disulphide bonds than normal endoplasmic reticula, which is
associated with the loss of protein folding regulation (116).
Interestingly, exercise reduces negative outcomes in Alzheimer’s disease, including
the accumulation of misfolded proteins (159). The unfolded protein response (UPR) is
induced within contracting muscle cells and plays an important role in cell protection from
stress, mainly through an essential chaperoning system (50). Exercise seems to improve
many ERS-related pathologies, such as diabetes mellitus, neurodegenerative diseases,
sarcopenia, or cardiovascular alterations (81, 91, 169) and PGC- the redox sensible
master gene involved in the beneficial effects of exercise interacts with ATF6a to regulate
the UPR in muscle (169). Many proteins included in the axis that regulates UPR activation
(e.g. PERK, eIF2a, ATF4, CHOP, IRE1, XBP1, BiP and ATF-6) are regulated in both muscle and
remote tissues following exercise training. For example, exercise training upregulates ER-
chaperones, such as binding immunoglobulin protein (BiP) in mouse muscle (39, 110), liver
(31) and brain (89, 96). In summary, both acute and chronic exercise seems to activate UPR
signaling in order to alleviate ERS-related pathologies (50).
A tight regulation of unfolded protein response is also crucial to the correctly
folding of tumour suppressor proteins (79, 166). An exercise-mediated control of tumour
suppressor proteins has been proposed to be one plausible mechanism for the lower
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incidence of cancer in regular physically active individuals (113, 166). Indeed, a tumour
suppressor protein might be delivered from contracting muscle or synthetized directly in
different tissues by ROS-mediated signaling that is induced by physical exercise (Figure 1A).
Another possible mechanism is related to decreased insulin resistance in trained
individuals, since insulin resistance seem to be positively related to tumour development
(3). Once again, notably, such hypotheses fit well with the association of being actively
healthy and the lower incidence of cancer (83).
4. Lipid Peroxidation as a mediator of crosstalk between different tissues

Over the last three decades, an extensive body of literature has shown the role of
lipid peroxidation, not only in the pathophysiology of diseases, but also in cell biology and
human health (9). Lipid peroxidation is a process in which oxidants attack lipids containing
carbon-carbon double bond(s) and involves hydrogen abstraction from a carbon, with
oxygen insertion resulting in lipid peroxyl radicals and hydroperoxides (27). Increased ROS
production can lead to membrane lipid peroxidation and different cell fate, depending on
the cellular metabolic state and repair ability (9).
Products of lipid peroxidation (at low rates) can mediate cell to cell communication
during exercise. NADPH oxidase 2 (NOX2) is expressed at the sarcolemma of skeletal
muscle, and it seems to be the main source of ROS during contractile activities that
produce superoxide in the extracellular medium (74, 77, 138). It is believed that
superoxide is rapidly converted into H2O2, which is more stable and membrane permeable,
acting as a signaling molecule. However, superoxide production near the sarcolema might
increase the oxidation of extracellular lipids/lipoproteins, converting linonelic acid into 13-
Hydroxyoctadecadienoic acid (13-HODE) and cholesterol into oxysterol, which is able to
activate transcription factors such as peroxisome proliferator-activated receptor gamma
(PPARy) and liver X receptor alpha (LXR-, respectively (41). 4-hydroxynonenal (4-HNE) is
another product of lipid peroxidation that can activate transcription factors. It was
demonstrated that 4-HNE activates Nrf2, increasing mitochondriogenesis and antioxidant
defence (95). Interestingly, Nrf2 is activated after physical exercise in several tissues
including the brain, kidney, and testis (44). Once products of lipid peroxidation are
increased in the blood after physical exercise (66), it is possible that they can communicate
with skeletal muscle and with other distant cells in the body (Figure 2).
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5. Metabolites acting as communicating molecules through ROS-dependent
mechanisms
A growing body of evidence suggests that different metabolites produced during

muscle contraction are secreted during physical exercise. Interestingly, these metabolic
products can act like chemical mediators between cells (20). Lactate is a very well-known
metabolite produced by glucose oxidation during muscle contraction that has been
suggested as a candidate molecule to communicate muscle with remote cells. Formerly, it
was believed that lactate was a waste product of glycolysis, even referred to be the cause
of fatiguing muscle during physical exercise (24). Lactate role has changed from a possibly
bad intermediate molecule in muscle physiology to an important messenger molecule
during physical exercise (1). Nowadays, many physiological functions are attributed to
lactate, such as slowing muscular fatigue (117), increasing force production (92) and
serving as a high energy rich carbon skeleton for many crucial biochemical processes
within cells (24, 140).

The biochemical aspect of lactate production and its physiological role were
extensively reviewed by Brooks in 2018 (24). In brief, there are three main proposed
functions for lactate, as follows: 1) as an energy source, 2) a major gluconeogenic
precursor following exercise, mainly due to a shuttle between the skeletal muscle and the
liver (the Cori cycle), and 3) a “lactormone”, able to mediate autocrine, paracrine and
endocrine communication (24). New information concerning a shuttle between
contracting muscle and microbiota has just become available, which suggests that
intestinal microbiota are able to metabolize lactate and thus influence the exercise
capacity (143). Besides, a potential mechanism showing that lactate might be able to
stimulate ROS-mediated signaling has also been proposed (Figure 3).
Intracellular lactate levels increase during physical exercise in an intensity
dependent way. Lactate can cross the sarcolemma and reach blood circulation through
monocarboxylate transporters (MCTs). Serum lactate can be uptaken by a wide range of
cells like contracting skeletal muscles themselves (16), heart (59), brain (132), liver (48),
kidney (112), astrocytes or neurons (126). It is believed that slow twitch skeletal muscle
fibers and liver are the most important tissues for the removal of lactate from blood (60)
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(Figure 3), and the expression of MCTs is crucial for the uptake of lactate from circulation.
Moreover, functional mitochondria are fundamental for lactate to be oxidized (12, 24).
As proposed by Hashimoto et al., lactate may act as a communicating mediator

called “lactormone” that couples contracting muscle to other tissues (69). Supporting this
hypothesis, intraperitoneal L-lactate administration was found to increase mitochondrial
content in diverse mouse tissues such as liver and brain (46), as well as PGC-1 and
uncoupling protein 3 (UCP3) expression in gastrocnemius (90). Because these events are
also observed after physical exercise, the transitory increase in blood lactate during
physical exercise can contribute to the widespread health adaptations that occur in
remote tissues. Recently, it was demonstrated that lactate was able to upregulate
transforming growth factor beta2 (TGF2) in white adipose tissue. Furthermore, TGF-2
was secreted by adipose tissue in response to physical exercise, which is related to the
improvement of glucose tolerance. Interestingly, the administration of a lactate-lowering
agent dichloroacetate during exercise training decreased circulating TGF-β2 levels and
reduced exercise-stimulated improvements in glucose tolerance (154).
A hypothetical model is proposed in figure 1, which shows the probable redox-
mediated mechanisms through which contracting skeletal muscle communicates with
remote tissues. ROS might be involved in lactate action in different tissues. Indeed, lactate
increases the expression of genes involved in lactate transport and mitochondrial
oxidation in a ROS-dependent manner (69), although the source of ROS has not been
defined. Recently, it has been shown that lactate-mediated increase in PGC1- expression
was blunted by N-acetyl cysteine pre-treatment in skeletal muscle (90, 115). Figure 3
illustrates the hypothetical sources of ROS that can be activated by lactate: mitochondria,
xanthine oxidase and NAD(P)H oxidase. During lactate oxidation, NADH is generated,
increasing mitochondrial oxidative phosphorylation, which can increase ROS production.
Another potential mechanism may be the use of NADH derived from lactate oxidation by
NAD(P)H oxidase to produce ROS. This hypothesis is supported by experiments in
Drosophila in which lactate stimulates intestinal NADH-dependent ROS production (82). A
dysbiosis marked by overgrowth of Lactobacillus plantarum in the fly gut increases lactate
production and promotes over proliferation of intestinal stem cells. Using this model, the
authors proposed that lactate is a key element for communication between microbes and
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intestinal cells, and that NADH produced by oxidation of lactate derived from bacteria
could be a substrate for NAD(P)H oxidase to increase ROS production (82). In this context,
it would be interesting to test whether the same process occurs following exercise.
Lactate also seems to exert a free radical scavenger function, able to act as an
antioxidant in some specific conditions as heart ischemia, since the perfusion with a
lactate solution protects the tissue from oxidative damage (65). These newly proposed
functions of lactate, acting as a free radical scavenger and as a signaling molecule are
illustrated in figure 3.
A purine cycle also intermediates the crosstalk between contracting muscle and
liver. Mostly in the liver, inosine and hypoxanthine derived from the muscle can be
converted into uric acid by xanthine oxidase (71). This cycle might be a potential
mechanism by which exercise promotes (paradoxically) a pro-oxidant effect due to
xanthine oxidase in the liver, and an increase of urate, a powerful antioxidant in circulation
(114). In fact, increased plasma total antioxidant capacity has been observed after high
intensity exercise (71). The circulating urate can be taken up by different cells and,
following a recovery period after exercise, converted into allantoin (73). During this
process, there is a scavenger of the hydroxyl radical, hydroperoxide, and lipid peroxidation
(37). Interestingly, urate is taken up preferentially by the contracting muscle, and not by
the resting ones (72). Although the mechanism is not well understood, urate might be
useful for protecting the contracting muscle from oxidative damage once its exogenous
administration reduces exercise-induced oxidative stress (165).
Based on this intimate co-operation between muscle and liver, we propose
synchronized and tightly regulated ROS-mediated signaling in remote tissues that might be
dependent on the products of muscle contraction and liver processing. This crosstalk is a
well-organized network that synchronizes contracting muscle and other remote tissues to
ensure optimal redox-mediated signaling during exercise and recovery (as illustrated in the
hypothetical schema in figure 1). For example, lactate is produced and released into blood
circulation, reaching multiples different tissues, then altering the metabolism and
promoting a ROS-mediated signaling. Simultaneously, intense ATP hydrolyses, mostly
found during intense exercise, promotes the release of inositol and hypoxanthine, which
mostly reaches its peak after exercise. Once in the liver, urate is produced from
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hypoxanthine and released into circulating blood, delivering a potent antioxidant and
probably counteracting the peaks of ROS production. This hypothetical possibility
elaborates the fine-tuning process required for the exchange of purines and lactates that
are derived from contracting muscle and is dictated by the intensity of exercise. When this
fascinating and natural communication is perturbed, a disaccord in redox-mediated
signaling can lead to impaired function in many tissues and blunt the widespread health
benefits of exercise.
6. H2O2 gradient as a chemoattracting agent supporting skeletal muscle regeneration

In skeletal muscle, cooperation between immune and muscular cells is observed

during muscular regeneration. The recruitment of neutrophils and monocytes from blood
circulation are mediated by chemoattracting molecules that are secreted by many cells
(e.g. endothelial cells, satellites cells and immune-resident cells) at the lesion site. Multiple
factors as and C-C motif ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and
tumour necrosis factor α (TNFα) are known to attract monocytes and to differentiate them
into macrophages respectively (128), orchestrating the process of myogenesis (32, 94,
137). H2O2 gradient also serves as a chemoattracting agent. In zebrafish model, a H2O2
gradient generated by the DUOX enzyme (after tissue injury) recruits leucocytes to the site
of the lesion to promote wound healing (118). Moreover, H2O2 production seems to be
crucial to promote progenitor cell proliferation during acute kidney lesions (33). In these
two examples, the inhibition of H2O2 generation abolished the regenerative process. In
mammals, the role of DUOX1 in the recruitment of immune cells was demonstrated in lung
inflammatory response, which was found to be crucial to the regenerative process (30, 67).
Thus, one can speculate that exercise induced-H2O2 generation in skeletal muscle cells
could create a gradient to attract supportive cells to participate in exercise responses.
Following eccentric exercise, there is a peak in pro-inflammatory CD68+
macrophages infiltration at 48 h (100, 102) that switches to anti-inflammatory CD163+
macrophages after 72h (57, 131). Given that ROS are produced and released into the blood
from skeletal muscle (11, 34, 84), one can hypothesize that these molecules participate in
the recruitment of immune cells to support cellular regeneration after damaging exercise.
In fact, DUOX1 is expressed in muscular progenitor cells and its expression declines during
myogenesis (139), suggesting that DUOX1 could be a source of ROS from satellites
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muscular progenitor cells to communicate to other cells through an H2O2 gradient, as
observed during zebrafish and mammalian lung and kidney injury.
7. Myokines and its ROS-dependent regulation - Communicating the contracting muscle

to the whole body
Following contractile activity, skeletal muscle cells increase the production and
release of several molecules such as cytokines or other small proteins (~5–20 kDa) and
proteoglycan peptides that show autocrine, paracrine and/or endocrine effects. Several
well-known cytokines (e.g. IL-6, IL-8, IL-10 IL-15 etc.) have been shown to be secreted by
skeletal muscle during physical exercise and, as a result, they are referred to as myokines
(123). Myokines are involved in exercise-associated metabolic changes and not only in
muscle, but also in other tissues like heart, adipose tissue, liver, pancreas bone, and brain.
Myokines play important roles in metabolic changes that are induced by physical exercise.
They also participate in tissue regeneration and repair, immunomodulation and cell
differentiation (124). The interconnections between contracting muscle and practically all
cells are complex and not a completely deciphered system. Currently, several myokines
have been described to be stimulated by physical exercise, including IL-6 (121), Irisin (19),
IL-15 (146), FGF21 (156), LIF (23), MCP1 (28). Interestingly, the PGC-1and Akt/mTOR
pathways seem to be involved in myokine secretion, and both of them are redox sensitive
intracellular pathways (Figure 4). The most well studied myokines are IL-6 and Irisin (121).
Skeletal muscle IL-6 secretion increases after a single bout of physical exercise
(124), which induces liver glucose output (52). IL-6 secretion during physical exercise is not
related to tissue damage or pro-inflammatory processes (124, 142, 150), and seems to be
regulated by intracellular calcium levels (167). It was demonstrated that the administration
of a cocktail of antioxidants containing Vitamins A, C and E (161) or Vitamin C and E (54)
before a single bout of physical exercise attenuated IL-6 secretion in humans. L-NAME, a
NOS inhibitor, also attenuated the increase in IL-6 mRNA levels in response to exercise
(151). Additionally, it has been suggested that NOX enzymes are involved in IL-6 secretion
by skeletal muscle during physical exercise, since apocynin, a NOX inhibitor, attenuated
this phenomenon, which was also occurred in a gp91phox knockout animal (75).
IL-6, secreted by contracting skeletal muscle during resistance training, seems to
activate satellite cells through the activation of STAT3, which is crucial for hypertrophy
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(144). It has not been investigated if this action is dependent on ROS-mediated signaling,
however, ROS can stimulate STAT3 via a modification on its Tyr 7055 and Cys 253 (147).
Furthermore, IL-6 stimulates ROS production in plenty of different cell types (163), so it is
possible that ROS can act as an intermediate in the effect of IL-6 in remote tissues during
exercise. For instance, increased insulin sensitivity and fatty acid oxidation that is
stimulated by IL-6 (26) might, in part, be dependent on the activation of AMPK by ROS
(157). Also, the stimulus of muscle glucose uptake following exercise might be through a
similar mechanism (61). The interplay between IL-6 and AMPK has also been confirmed in
different tissues (88).

Irisin was first described as a myokine with the ability to induce a browning
phenotype in white adipose tissue, raising basal metabolic rates (19) (Figure 5). Several
studies showed that irisin promotes widespread effects in remote tissues, preventing
oxidative stress and tissue damage. Nrf2 activates a well-known antioxidant pathway that
protects against various pathological conditions (43, 97, 99), and its activation seems to
correlate with the anti-fibrotic and anti-oxidative effects of irisin (33, 45, 171). For
instance, irisin treatment protects hepatocytes in vitro from overproduction of ROS that is
induced by H2O2 (13). Moreover, irisin mediates part of the protective effect of
dexmedetomidine in livers under intestinal ischemia/reperfusion damages via decreasing
inflammasome markers and ROS production (51). Additionally, irisin is known to decrease
several pro-inflammatory cytokines and increase antioxidant defences (e.g. SOD and GPX)
in hepatocytes through Nrf2 upregulation (45). In endothelial cells, irisin stimulates
angiogenesis via AKT/mTOR/S6K1/Nrf2 signaling, which reduces the higher ROS content
induced by oxidized LDL (171) (Figure 5).
A major role of oxidative stress in fibrosis development is caused by over-activated
Smad-3 signaling (18) that leads to exacerbated ROS production (14). Mechanistically,
irisin-induced Nrf2 activation in cardiac cells suppresses the ROS generation that is induced
by angiotensin II, which is triggered by TGF-β1 in fibroblasts (33). Moreover, irisin was
found to prevent heart remodelling (98) and reduced blood pressure of spontaneously
hypertensive rats through endothelial cells oxidative stress reduction via AMPK and NO
signaling (56). In a type 2 diabetes mice model, irisin ameliorated endothelial function by
reducing the overproduction of superoxide and peroxynitrite (172). Yet, in endothelial
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cells, irisin also reverted the oxidative stress induced by advanced glycation end products
by decreasing eNOS activity and therefore, preventing the endothelial damage that is
secondary to inflammasome overactivation (40).

In macrophages, irisin was found to increase proliferation and phagocytosis ability
(105) and protected these cells against oxidative stress damage via upregulation of
antioxidant defences, even under LPS stimulation (106). Given the undeniable implication
of macrophages in the development and progression of many diseases, a direct action of
exercise-induced irisin on macrophages could contribute to the widespread beneficial

effects of exercise. Some of these effects are illustrated in figure 5.

Previous studies have suggested that myokines mediate the beneficial effects of
exercise in brain function (Figure 5). Under an ischemia/reperfusion stroke, brain cells
experiment a glucose/oxygen deprivation that triggers an intense ROS production that can
lead to an irreversible injury. Using in vitro model, irisin protected neuron cells from
oxidative stress via decreasing the higher ROS production and inflammation through
increased SOD expression, consequently protecting neurons from stroke-induced
apoptosis (127).
In 1944, one of the first reports suggested that exercise reduced tumour growth
and progression in mice (136). Since then, there are several evidences showing that a
healthy lifestyle hinders the development of several types of cancer and regular exercise
can protect from a large range of diseases (49, 119, 134). However, the precise
mechanisms are still unclear. Regarding the effect of exercise on tumour growth, there are
many conflicting results that have been obtained from rodent experiments. Recent studies
have shown that exercise may decrease tumour growth if tumour-bearing mice trained 4
weeks prior to inoculation with B16 melanoma cells (125), while another study suggested
that avoiding free access to exercise because they observed increased tumour growth and
decreased life span in a model of intramuscular tumour inoculation (8). Despite the gap of
knowledge about the mechanisms of actions involved in this process, regular exercise has
been recommended as a supplementary therapy for cancer patients (83). Based on
existing evidence, some public health organizations have issued physical activity guidelines
also for cancer prevention. Regarding the large number of studies conducted on physical
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activity and cancer prevention, most of them have an incomplete description of physical
activity (such as type, intensity, duration and metabolic responses).
Mechanistically, an anti-proliferative effect of serum collected from mice after

moderate exercise intensity (50% of maximal exercise capacity), but not from mice
submitted to high intensity swimming exercise (50 and 80% of maximal capacity,
respectively), confirms the idea that a mix of myokines are secreted that are able to affect
tumour cells, reducing tumour growth (2). However, another study showed that high
intensity training (corresponding to 85% of VO2 max) was able to promote a reduction in
tumour mass (10). By screening 28 potential candidate molecules upregulated in serum

after swimming exercise, Oncostatin M has emerged as a candidate for reducing mammary
cancer cells growth (78). Another candidate able to prevent the initiation of a tumour was
the “Secreted Protein Acidic and Rich in Cysteine” (SPARC). The level of circulating SPARC
increased in mice and humans following a single bout of exercise. Low intensity exercise
induces SPARC expression and secretion that reduces the formation of aberrant crypt foci
and prevents the onset of colon cancer (6). SPARC expression seems to be induced by low
and moderate exercise programs (149) and is mainly secreted by slow twitch fibers instead
of fast twitch fibers following resistance exercise (148) (Figure 5). Apart from its possible
direct action as an anti-tumor molecule, SPARC also seems to mimic some of the beneficial
effects of exercise on glucose metabolism via AMPK activation (5).
Therefore, myokines secreted by the muscles may mediate the beneficial effects of
exercise on tumour development. Although it is a promising and innovative theme, the
effects of contracting muscle myokines (e.g. IL-6, Irisin, FGF21, Oncostatin M, SPARC and
etc) on tumour growth and progression using xenograph and inoculated rodent models are
still relatively unknown.
8. Extracellular vesicles released following exercise and physiological perspectives
Another important product secreted by skeletal muscle during contraction are
extracellular vesicles that include exosomes and microvesicles (also known as shedding
vesicles, ectosome). Exosomes are complex 20–100 nm vesicles formed by the inward
budding of endosomal membranes to form large multivesicular bodies. These vesicles are
released out of the cell when the multivesicular body fuses with the plasma membrane.
The exosome is characterized by soluble and membrane-bound proteins, lipids,
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metabolites, DNA, and RNA (mRNA, miRNAs, and other small regulatory RNAs) involved by
a protective lipid bilayer that can modify signaling pathways and protein expression in
remote tissues (160).

Physical exercise considerably increases the metabolic demands of the body and
the vesicle trafficking of metabolic mediators might be a tool through which tissues can
share resources during this physiological situation. Using quantitative proteomic
techniques and intravital imaging experiments, Whitham et al. (2018) demonstrated that
skeletal muscle contraction stimulates extracellular vesicle release and provides the means
for tissue crosstalk during exercise (168). Although the temporal aspects of exosome and
small vesicle biogenesis and transport in exercise are unknown, it has been demonstrated
that the release of exosomes is associated with increases in the levels of intracellular
calcium (141). Because motor neuron stimulation of skeletal muscle fibers leads to a rapid
release of Ca2+ from the sarcoplasmic reticulum (109), it is plausible that a transient Ca2+
increase during skeletal muscle contraction is likely involved in exosome release.

ROS seems to be involved in the secretion of extracellular vesicles since pro-oxidant
conditions seem to induce their release (15). Additionally, NADPH oxidase and nitric oxide
synthase-2 (NOS-2) inhibitors reduced the production and release of neutrophil
microvesicles (158). Furthermore, tumour and senescent cells have altered redox balances
with elevated ROS levels, which is related to the higher number of microvesicles secreted
by these cells (29, 80). From an exercise perspective, more research is necessary to
elucidate the relationship between increases in ROS production during skeletal muscle
contraction and microvesicle release (Figure 1). However, some evidence suggest that
disrupted ROS-mediated signaling caused by NAD(P)H oxidase inhibition (76), or aging,
through exacerbated ROS generation (107), can affect the nature of exosomes (e.g. Heat
shock proteins expression) delivering and potentially hampering the widespread beneficial
effect of exercise.
Heat shock proteins (HSPs), for example, can be transferred between two cells
(170) and participate in the maintenance of protein homeostasis (155). Previous studies
suggested that Hsp70 secretion is mediated by ATP-binding cassette transporter (ABCA1)
transmembrane transporters (4). Furthermore, three other mechanisms can also be
related to Hsp70 release, such as: 1) cell lysis with consequent release of cytoplasmic
Page 17 of 45
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content; 2) cell surface blebbing and Hsp70 release in microvesicles to the extracellular
fluid; and 3) through endolysosomes (58, 103, 104). HSPs are molecular chaperones that
facilitate protein folding and maintain protein structure and function during cellular stress.
HSPs are involved in a wide range of cellular processes that are perturbed by oxidative
stress, such as protein folding, apoptosis, and inflammation (85, 86). As recently discussed
(108), the intercellular communication via exosomes might compensate for the imbalance
in HSP levels in almost every cell (155). For example, differentiated neurons seem to be
dependent on a transfer of HSP from other cells to maintain their cytoprotection against
stress (101). It has been demonstrated that intercellular HSP transfer occurs by exosomes
following exercise (93, 164), raising the idea that this intercellular communication is critical
to ensure an integrated stress response across different tissues (120). The exosomes
secreted after acute exercise have higher levels of HSPs (55, 168). In an opposite way,
muscle denervation alters the content of exosomes (38), suggesting a crucial role for
contractile activity on exosome content and secretion.

Alzheimer’s is a neurodegenerative disease in which there is a prevalent protein
folding disorder in the brain, the heat shock protein response in some neurons becomes
reduced (25). It has been extensively demonstrated that HSP is involved in protein
homeostasis in the brain, and an accumulation of misfolded proteins (amyloid peptides)
contributes to the progression of this neurodegenerative disease (7). Interestingly, an
increase in HSP70 expression improves cognition function and reduces amyloid- peptide
levels in Alzheimer's model mice (152). Based on the abovementioned mechanisms, it is
tempting to suggest that HSP transfer through exosomes, which follows exercise, could
retard or even prevent the development of brain diseases; and this hypothesis is
illustrated in Figure 5.
9. Conclusions
The widespread beneficial effects of physical exercise are mediated by multiple

soluble factors that interfere with a variety of signaling pathways, and the decoding of
these pathways is a challenge for basic and translational research. Fascinatingly, optimal
ROS-mediated signaling is necessary to achieve the beneficial effects of exercise. In a
Page 18 of 45
18
common way, exercise-secreted mediators seem to operate via PGC-1 and Nrf2, two
redox sensible pathways that promote healthier conditions globally.
Recently, attention has focused on the mechanisms of action of new myokines.

Many exercise responses and adaptations may be dictated by the (un)balance of
“cytokines cocktail”, defined as a net balance between deleterious hampering molecules
versus benefit enhancing molecules. Understanding how aging and obesity blunt some of
these exercise adaptations and proposing a multiple and combined therapeutic strategy
that will optimize the beneficial effect of exercise in many diseases is a critical issue in
exercise biology. In addition, the ROS-rich environment that is created after exercise is not
exclusively restricted to contracting muscle. Understanding the processes by which the
beneficial effects of exercise occurs is, therefore, a prerequisite to defining how lack of
exercise contributes to a poor quality of life and high incidence of disease. Therefore,
further studies designed to decipher the mechanism of action of myokines are
indispensable to understand the redox code that underlies the beneficial effects of
exercise, and thus, will expand the therapeutic strategies for many diseases.
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List of Abbreviations
13-HODE- 13-Hydroxyoctadecadienoic acid

4-HNE - 4-hydroxynonenal
AMPK - AMP-activated protein kinase
CAMK -Ca2+/calmodulin-dependent protein kinases
DUOX – Dual oxidase
ER - Endoplasmic reticulum
ERS - Endoplasmic reticulum stress
FGF21 - Fibroblast growth factor 21

GLUT4 – Glucose Transporter 4
H2O2 Hydrogen Peroxide
HSPs - Heat shock proteins
IL - Interleukin
iNOS – inducible Nitric oxide synthase

LIF - Leukaemia inhibitory factor
L-NAME - N(G)-nitro-L-arginine methyl ester
LXR-Liver X receptor alpha
MAPK - Mitogen-activated protein kinase
MCP1 - Monocyte chemoattractant protein 1
NADH – Nicotinamide adenine dinucleotides
NF-B - nuclear factor-kappa B
NOX – NADPH oxidase
Nrf2 – Nuclear factor erythroid 2-related factor 2
O2•- superoxide
OH• - Hydroxyl
PDK4 – Pyruvate dehydrogenase kinase 4
PGC-1-Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
PPARy - Peroxisome proliferator-activated receptor gamma
TGF- transforming growth factor-beta
UCP3 - Uncoupling protein 3
Page 20 of 45
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Acknowledgments
The study was supported by research grants from CNPq, Fundaçao Carlos Chagas Filho de
Amparo a
Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES). The authors would like to thank Sapiens scientific
illustrations for the design of the figures.
Author Contributions
All authors listed have made a substantial, direct and intellectual contribution to the work,
and approved it for publication. R A Louzada, J Bouviere and L Matta wrote the paper; DP
Carvalho, C Dupuy and JP Werneck-de -Castro revised the paper; R A Louzada and R S
Fortunato designed the outline of the paper and wrote the paper.
Page 21 of 45
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Figure legends

Figure 1. Exercise-induced reactive oxygen species (ROS) waves illustration.

A) Contracting muscle creates an oxidative environment through consecutive reactive
oxygen species (ROS) spikes. The first ROS wave is produced mainly by skeletal muscle
NADPH oxidase (Nox2), induces activation of ROS-mediated signaling pathways, and
promotes correct folding of newly born proteins. Skeletal muscle releases signals to
remote tissues in many ways during muscle contraction. For example, metabolites
released by skeletal muscle can be taken up and metabolized in remote tissues, an event
that can stimulate ROS production. Moreover, myokines can also control the production of
ROS in remote tissues. Products of lipid peroxidation (13-hydroxyoctadecadienoic acid
[HODE] and 4-hydroxynonenal [HNE]) produced during skeletal muscle contraction are
capable of binding to nuclear receptors in remote cells in which these compounds can then
regulate gene expression. Finally, exosomes released by the muscles undergoing
Page 41 of 45
41
contraction might stimulate signaling pathways in remote tissues. ROS production in
remote tissues following contraction is proposed as a second ROS wave.
B) First and second ROS waves induced by physical exercise in a temporal perspective.
During skeletal muscle contraction, the first ROS wave within skeletal muscle occurs during
exercise and can spread to remote tissues. At the end of exercise, while contracting muscle
causes a decrease in ROS production, remote tissues present the second ROS wave,
leading to activation of ROS-mediated signaling pathways.

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42

Figure 2. Lipid peroxidation as a mediator of skeletal muscle–remote tissue crosstalk.

NADPH oxidase is proposed as the main source of ROS during contractile activity and due
to its transmembrane location, ROS production is very close to the phospholipids of the
membrane. Circulating lipids and membrane phospholipids can be attacked by
extracellular-produced ROS to form lipid peroxidation intermediates that can interact with
nuclear receptors in remote tissues and modulate gene expression.
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43
Figure 3. Crosstalk between muscle and other tissues. Skeletal muscle contraction
increases glucose oxidation rates in an exercise intensity-dependent manner and
stimulates the conversion of pyruvate into lactate via lactate dehydrogenase (LDH).
Lactate is mainly produced by fast-twitch muscles during exercise and is released muscles
undergoing contraction to the blood circulation through specific transporters (such as
monocarboxylate transporters [MCT]). Once in circulation, lactate can be taken up by
many tissues, such as liver, white adipose tissue (WAT), brain, heart, and kidney. In the
liver, lactate is converted into pyruvate by pyruvate dehydrogenase (PDH), causes an
increase in the levels of NADH+, and stimulates several intracellular metabolic pathways.
Robust adenosine triphosphate (ATP) hydrolysis produces adenosine diphosphate (ADP),
which is converted into ATP and adenosine monophosphate (AMP) by adenylate kinase
(AK). AMP deamination yields hypoxanthine, which is released into the circulation.
Xanthine oxidase converts hypoxanthine into urate and also produces superoxide, leading
to redox-mediated signaling in the liver. Urate is a potent antioxidant molecule that is
released into the circulation. The proposed crosstalk between muscle and liver involves
spatially and temporally synchronized ROS production and ROS scavenging to ensure that
correct ROS-mediated signaling occurs within the whole body during and after exercise. In
the left panel, intestinal NADH-dependent ROS production is proposed as a mechanism
that may use NADH derived from lactate oxidation by NAD(P)H oxidase to produce ROS in
a microbiota-dependent way.
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Figure 4. Myokines and ROS dependence. The most studied myokines are illustrated and
its ROS dependence is highlighted. Interleukins (IL)-6, -8, and -15 are regulated by ROS. The
control of several myokines relies on the activation of human peroxisome proliferator-
activated receptor γ coactivator-1 alpha (PGC-1) and protein kinase B/mechanistic target
of rapamycin (AKT/mTOR) signaling, but its dependence on redox signaling has not
completely been investigated yet.

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Figure 5. Crosstalk between skeletal muscle and other tissues. The regular practice of
physical exercise promotes widespread health benefits that can prevent and treat many
diseases. Most of the desirable effects of physical exercise are mediated by myokines that
can facilitate communication of contracting muscles with remote tissues. The mechanism
of action of many myokines rely directly or indirectly on redox signaling.

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Antioxidants and Redox Signaling

REDOX SIGNALING IN WIDESPREAD HEALTH BENEFITS OF

Ruy A. LOUZADA1,2*, Jessica BOUVIERE1, Leonardo P. MATTA1, Joao Pedro S. WERNECK-DE-

to promote the benefits of exercise through paracrine or endocrine signaling. This

released from skeletal muscle in response to intracellular ROS stimulated by physical

factors, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha

to an accumulation of unfolded or misfolded proteins inside the endoplasmic reticulum

4. Lipid Peroxidation as a mediator of crosstalk between different tissues

A growing body of evidence suggests that different metabolites produced during

within cells (24, 140).

As proposed by Hashimoto et al., lactate may act as a communicating mediator

6. H2O2 gradient as a chemoattracting agent supporting skeletal muscle regeneration

In skeletal muscle, cooperation between immune and muscular cells is observed

7. Myokines and its ROS-dependent regulation - Communicating the contracting muscle

different tissues (88).

secondary to inflammasome overactivation (40).

exercise-induced irisin on macrophages could contribute to the widespread beneficial

effects of exercise. Some of these effects are illustrated in figure 5.

Mechanistically, an anti-proliferative effect of serum collected from mice after

tumour mass (10). By screening 28 potential candidate molecules upregulated in serum

remote tissues (160).

increase during skeletal muscle contraction is likely involved in exosome release.

contractile activity on exosome content and secretion.

The widespread beneficial effects of physical exercise are mediated by multiple

Recently, attention has focused on the mechanisms of action of new myokines.

13-HODE- 13-Hydroxyoctadecadienoic acid

FGF21 - Fibroblast growth factor 21

iNOS – inducible Nitric oxide synthase

Science 305: 1112–1113, 2004.

Physiol 107: 261–265, 2009.

related vesicles. Mol Biol Cell 10: 1463–1475, 1999.

Cell Longev 2014: 360438, 2014.

generation of reactive oxygen species. Biochimie 89: 1464–1473, 2007.

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REDOX SIGNALING IN WIDESPREAD HEALTH BENEFITS OF EXERCISE (DOI: 10.1089/ars.2019.7949)

REDOX SIGNALING IN WIDESPREAD HEALTH BENEFITS OF EXERCISE (DOI: 10.1089/ars.2019.7949)

Figure 1. Exercise-induced reactive oxygen species (ROS) waves illustration.